This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ***Please note: Dr. Boyson re-joined COBRE as a project PI as of 9/1/2009. NKT cells comprise an innate-like T cell subset that is notable for its ability to rapidly activate and/or modulate function of leukocyte subsets of the innate arm of the immune system. NKT cells can be activated through recognition of microorganism-derived glycolipids presented by CD1d (direct pathway), or through IL-12 and IL-18 produced by antigen presenting cells activated by TLR ligands (indirect pathway). Upon activation, NKT cells rapidly secrete large amounts of a wide variety of cytokines and activate other leukocyte subsets such as dendritic cells, macrophages, B cells, and natural killer cells. Therefore, NKT cells are uniquely poised to influence early events in the developing immune response. We have shown that 129X1/SvJ and 129S1/SvImJ NKT cells are severely deficient in IFN-g, IL-4, and TNF production in response to the prototypical NKT cell agonist a-galactosylceramide. Furthermore, they exhibit significantly lower numbers of liver, but not spleen or thymus, NKT cells. Since macrophage TNF production is impaired in NKT cell-deficient B6 mice, we asked whether impaired NKT cell number and function in 129 mice would result in impaired macrophage function after an in vivo LPS challenge. We found that the two 129 strains, as well as other inbred strains of mice deficient in NKT cell number and function, exhibit a severe deficiency in serum TNF production in response to an in vivo challenge with the TLR4 ligand, lipopolysaccharide. Intracellular cytokine staining demonstrated that low serum TNF levels were due to significantly impaired monocyte and macrophage TNF production in response to LPS. Strain-dependent differences in macrophage TNF production were not macrophage-intrinsic, suggesting that strain-dependent differences in macrophage TNF production after LPS challenge was due to in vivo regulation. Adoptive transfer of B6 NKT cells to the H2-matched 129 strains increased macrophage TNF production in response to an in vivo LPS challenge. Using a B6.129 congenic strain, we found that the locus controlling impaired macrophage TNF production in response to in vivo LPS challenge coincides with the Slam locus, which has previously been demonstrated to control NKT cell number and function in NOD mice. Based on these preliminary data, our central hypothesis is that genetic regulation of macrophage TNF production by Nkt1 occurs through the control of NKT cell number and function. To test this hypothesis, we propose to 1) Identify the genes in the Nkt1 locus that underlie the coordinate regulation of the in vivo response of macrophages to LPS and of NKT cell number/function by generating high-resolution congenic lines spanning the Nkt1 congenic interval, and 2) to investigate the mechanisms through which Nkt1 regulates NKT cell and monocyte/macrophage homeostasis and function. Aim 1: In the current funding period (08/09 [unreadable]04/10) we have constructed a high-resolution map of the B6.129-Slam congenic interval using SNP genotyping. Using these data, we have chosen appropriate microsatellite markers at the centromeric and telomeric ends of the interval for use in screening recombinants in backcross progeny. We have set up (B6.129-Slam X B6)F1 matings and we have begun to screen our first litters. Screening will be performed at the DNA Analysis facility at UVM. In addition, we have compared the transcriptional profiles of liver NKT cells from B6 and B6.129-Slam mice using microarray. This work done in collaboration with the Microarray and Bioinformatics Core, has resulted in the identification of a number of candidate genes within the congenic interval that could explain the observed phenotypes, including members of the Slam receptor and Ifi200 gene families. At present, we are confirming expression data on a limited number of candidate genes using QPCR. In the coming year, we plan on generating subcongenic lines which will be assessed for phenotypic differences in the NKT cell and macrophage compartments. We also plan on comparing transcriptional profiles of B6 and B6.129-Slam macrophages after in vivo LPS challenge. These studies will be conducted in collaboration with the Microarray and Bioinformatics Core. Aim 2: Our preliminary data indicate that liver NKT cell number and NKT cell cytokine production in response to in vivo aGalCer challenge is regulated by a gene(s) within the Slam locus. In the current funding period, we have begun to address the mechanisms underlying these phenotypes. Examination of liver NKT cell homeostasis using in vivo BrDU labeling and TUNEL staining revealed a significantly higher level of apoptosis in 129 liver NKT cells versus B6, and that a significant portion of this phenotype was regulated by the Slam locus. In the coming year, we will confirm and then extend these data by assessing liver NKT cell homeostasis in mixed B6 and B6.129-Slam bone marrow chimeras. In addition, we have begun to evaluate the role of Slam family receptors on NKT cell cytokine production. Many of the Slam family genes in the congenic interval exhibit numerous polymorphisms that define haplotypic differences between B6 and 129X1/SvJ mice. Our preliminary analysis revealed differential expression of many of these receptors on NKT cells, including the differential expression of Slamf6 (Ly108) alternative splice isoforms. Addition of anti-SLAMF6 mAbs to sorted NKT cells resulted in significantly impaired IFN-g production. In the coming year, we will confirm and extend these data using a panel of anti-SLAM receptor mAbs to assess their role in NKT cytokine production.